1. Field of the Invention
This invention relates to a nitric monoxide metabolite-polyoxyalkylene-hemoglobin complex which is useful in blood substitutes and organ perfusion solutions, a method for the production of said complex and an oxygen carrier solution which contains said complex, more particularly to a cell-free hemoglobin-based oxygen carrier which has high safety in blood and does not cause problems such as hypertension (blood pressure increase) at the time of injection into the living body.
2. Background Art
Currently, attempts are made positively to use cell-free hemoglobin extracted from human or bovine erythrocytes as an oxygen carrier substitute of erythrocyte blood transfusion.
It is necessary to solve various problems in order to use cell-free hemoglobin directly as an oxygen carrier substitute for erythrocyte blood transfusion. Firstly, stroma as a membrane fragment remains in erythrocyte hemolysate and causes blood coagulation. In this field, Rabiner et al. have established in 1967 a method for the production of stroma-free hemoglobin in which stroma is removed from a hemoglobin solution (Rabiner S. F. et al., Evaluation of a stroma-free hemoglobin solution for use as a plasma expander, J. Exp. Med., 126, 1127-1142, 1967), which solved the problem of disseminated intravascular coagulation (DIC) almost completely. Next, hemoglobin molecules are leaked from the renal glomerulus and exert toxicity upon the renal tube. The problem of renal toxicity has also been solved almost completely based on the finding by Bunn et al. in 1969 that excretion of hemoglobin from the glomerulus can be avoided by introducing intramolecular crosslinking into hemoglobin (Bunn F. et al., The renal handling of hemoglobin, J. Exp. Med., 129, 909-924, 1969) and the subsequent finding that excretion of hemoglobin into urine can be prevented and its intravascular half life can be prolonged by increasing molecular weight of hemoglobin through its chemical modification with polyethylene glycol (e.g., JP-B-5-64128 and JP-B-6-76333; the term xe2x80x9cJP-Bxe2x80x9d as used herein means an xe2x80x9cexamined Japanese patent publicationxe2x80x9d). Based on these technical improvement, studies on the cell-free hemoglobin as an erythrocyte substitution oxygen carrier have been advanced, and several products are already entering into clinical tests in the United States.
However, as the studies on such hemoglobin derivatives progressed, two new problems have been found. That is, blood pressure increasing reaction accompanied by vasoconstriction and abdominal pain accompanied by intestinal constriction.
It is considered that the main cause of the blood pressure increasing reaction as the first problem is vasoconstriction, particularly of the arteriole, induced by the administration of hemoglobin derivatives. This phenomenon is considered to be undesirable, because it is not recognized when normal erythrocyte transfusion is carried out, and constriction of the arteriole inhibits the flow of blood into capillary vessels where transfer of oxygen is carried out. It is considered that this vasoconstriction occurs due to the reaction of an endothelium-derived relaxing factor [EDRF: considered to be nitric monoxide or a nitric monoxide releasing substance (I. Sakuma, NO as a blood vessel relaxing factor, Experimental Chemistry, 9, 1347-1351, 1991)] with cell-free hemoglobin, which causes elimination of EDRF and thereby entails constriction of blood vessels (Motterlini R. et al., Hemoglobin-nitric oxide interaction and its implications, Transfusion Med. Rev., 10, 77-84, 1996). Particularly, it is important that EDRF is disappeared inside the blood vessel wall by the incorporation of hemoglobin molecules into the vessel through endothelium gaps (Nakai K. et al., Permeability characteristics of hemoglobin derivatives across cultured endothelial cell monolayers, J. Lab. Clin. Med., 132, 313-319, 1998).
Next, with the commencement of clinical tests in the human body in recent years, intestinal constriction caused by cell-free hemoglobin derivatives has been drawing attention as a new problem. Though its cause is not specified yet, this phenomenon is observed mostly in clinical tests of intramolecular crosslinking type hemoglobin, and it is assumed that its cause is the elimination of nitric monoxide as a candidate of anti-adrenergic anti-cholinergic neurotransmitter, also caused by the cell-free hemoglobin leaked for example from the endothelium (Murray J. A. et al., The effects of recombinant human hemoglobin on esophageal motor functions in humans, Gastroenterology, 109, 1241-1248, 1995).
Since the elimination of nitric monoxide by hemoglobin is the cause of each of these side effects, it has been proposed that it is effective to use a nitric monoxide metabolite-bonded hemoglobin to which transferring and releasing abilities of the nitric monoxide metabolite are added (Stamler, WO 96/30006). The nitric monoxide metabolite-bonded hemoglobin is a modified form of hemoglobin in which a nitric monoxide metabolite is reversibly bound to the xcex2-chain cysteine residue of hemoglobin, which can release the nitric monoxide metabolite having a physiological activity by compensating the elimination of nitric monoxide by the heme of hemoglobin. However, since this nitric monoxide metabolite-bonded hemoglobin is an intramolecular crosslinking type hemoglobin having small molecular weight, it still has a problem in that its half life in blood vessels is short due to its aptness to be excreted into urine.
Thus, the modified forms of hemoglobin are markedly promising as oxygen carriers for use, for example, in erythrocyte substitution blood transfusion and organ perfusion solution, but, as described in the foregoing, nothing has been proposed yet on their counterpart which is not easily be excreted, has therefore a long half life in the body and shows no side effects such as vasoconstriction and intestinal constriction.
Accordingly, great concern has been directed toward the development of a cell-free hemoglobin derivative which can be used safely and freely and is storable for a prolonged period of time.
An object of the present invention is to provide an oxygen carrier capable of being used safely and freely and being stored for a prolonged period of time, whose leakage from the renal glomerulus and vascular endothelium is extremely limited and which has a nitric monoxide metabolite releasing ability that can compensate removal of endothelium-derived nitric monoxide. Another object of the present invention is to provide an oxygen carrier solution which contains a specified amount of the nitric monoxide metabolite-polyoxyalkylene-hemoglobin complex (to be referred sometimes to as xe2x80x9cmodified compoundxe2x80x9d hereinafter) of the present invention. Since the oxygen carrier solution can be prepared simply and easily, it is useful as an erythrocyte substitution blood transfusion or organ perfusion solution.
The inventors of the present invention have conducted intensive studies on an oxygen carrier which can be used safely and freely causing no side effects such as renal toxicity, hypertension and abdominal pain and found as a result of the efforts that a derivative having a molecular weight of from 100,000 to 2,000,000, prepared by binding a specified polyoxyalkylene derivative and a nitric monoxide metabolite to cell-free hemoglobin extracted from human or bovine erythrocytes, is a markedly promising oxygen carrier, thus resulting in the accomplishment of this invention.
Accordingly, an object of the present invention is to provide a nitric monoxide metabolite-polyoxyalkylene-hemoglobin complex (modified compound) having a molecular weight of from 100,000 to 2,000,000 daltons, in which a polyoxyalkylene derivative is bound to from 10 to 30% in total of bindable amino groups in hemoglobin and a nitric monoxide metabolite is bound to from 10 to 100% in total of thiol groups of cysteine residues.
More particularly, it is the just described nitric monoxide metabolite-polyoxyalkylene-hemoglobin complex, wherein it uses a polyoxyalkylene derivative of formula (1): 
wherein B represents residue of a compound having from 2 to 6 hydroxyl groups, AO represents an oxyalkylene group having 3 or 4 carbon atoms, R represents a hydrocarbon group having from 1 to 30 carbon atoms or a hydroxyl group, k and m are numbers which satisfy 0xe2x89xa6kxe2x89xa6500 and 0xe2x89xa6mxe2x89xa6500, respectively, and also 20xe2x89xa6k+mxe2x89xa61000, as the average addition mol number of oxyethylene groups, l and n are numbers which satisfy 0xe2x89xa6lxe2x89xa610 and 0xe2x89xa6nxe2x89xa610, respectively, and also 0xe2x89xa6l+nxe2x89xa610, as the average addition mol number of oxyalkylene groups, a and b are numbers which satisfy 0xe2x89xa6axe2x89xa66 and 1xe2x89xa6bxe2x89xa66, respectively, and also 2xe2x89xa6a+bxe2x89xa66, and X represents a functional group capable of binding to the amino group shown by formula (2), (3), (4) or (5);
xe2x80x94(CH2)cxe2x80x94COOYxe2x80x83xe2x80x83(2)
wherein c is a number of from 0 to 2 and Y represents hydrogen or p-nitrophenyl group or N-hydroxysuccinimide residue,
xe2x80x94OCxe2x80x94(CH2)dxe2x80x94COOYxe2x80x83xe2x80x83(3)
wherein d is a number of from 2 to 6 and Y represents hydrogen or N-hydroxysuccinimide residue,
xe2x80x94(CH2)exe2x80x94CHOxe2x80x83xe2x80x83(4)
wherein e is 1 or 2, and
xe2x80x94COZxe2x80x83xe2x80x83(5)
wherein Z represents imidazole group.
Another object of the present invention is to provide the aforementioned nitric monoxide metabolite-polyoxyalkylene-hemoglobin complex, wherein it uses an s-nitroso-low molecular weight (preferably having 1,000 daltons or less) thiol compound such as s-nitrosoglutathione as the nitric monoxide metabolite.
A further object of the present invention is to provide an oxygen carrier solution which contains the nitric monoxide metabolite-polyoxyalkylene-hemoglobin complex in an amount of from 10 g/L to 200 g/L.
Yet another object of the present invention is to provide a method for producing a nitric monoxide metabolite-polyoxyalkylene-hemoglobin complex having a molecular weight of from 100,000 to 2,000,000 daltons, in which a polyoxyalkylene derivative is bound to from 10 to 30% in total of bindable amino groups in hemoglobin and a nitric monoxide metabolite is bound to from 10 to 100% in total of thiol groups of cysteine residues, which comprises carrying out a one step method, namely, allowing a polyoxyalkylene-hemoglobin complex to react with a nitric monoxide metabolite.
A still further object of the present invention is to provide a method for producing a nitric monoxide metabolite-polyoxyalkylene-hemoglobin complex having a molecular weight of from 100,000 to 2,000,000 daltons, in which a polyoxyalkylene derivative is bound to from 10 to 30% in total of bindable amino groups in hemoglobin and a nitric monoxide metabolite is bound to from 10 to 100% in total of thiol groups of cysteine residues, which comprises carrying out a two step method, namely allowing a polyoxyalkylene-hemoglobin complex to react with a nitric monoxide metabolite and then with a polyoxyalkylene derivative.